Project Summary: Human cardiac cells derived from human pluripotent stem cells (hPSCs) hold the potential to dramatically change the treatment of cardiac diseases and provide new strategies for rebuilding the adult heart after injury. In order to realize the therapeutic application of these cells, however, we need to better understand the mechanisms underlying the immune rejection during allogeneic cell transplantation. Here, the investigators propose a novel paradigm for control of hPSC immunogenicity, using recent advances in genome editing (CRISPR-Cas9) to program universal immune protective hPSC clones. These universal donor stem cells can then differentiate into human ventricular progenitors (HVPs) for allogeneic transplantation. The investigator recently developed a new efficient HVP differentiation method and performed extensive in vivo studies of HVPs and showed that the HVPs are better than cardiomyocytes in terms of survival, proliferation and generation of vascularized tissue. In this proposal, the investigator will employ recent advances in understanding the interaction between the human immune cells and transplanted donor cells and the CRISPR-Cas9 technique to design universal donor stem cells that confer immune protective properties for allogeneic transplantation. The investigator will use Cas9 to engineer stem cells for avoiding T-cell rejection via a gene knockout of beta2- microglobulin, which is essential for forming functional HLA-I molecules on the cell surface. In addition, a safe-harbor knockin for overexpression of PDL1 and CTLA4-Ig genes, which play critical T-cell immune modulation roles in allotransplantation, is also considered. Furthermore, to avoid the NK-mediated lysis of donor cells, the investigator will express HLA-E-B2M and HLA-G-B2M to inhibit the CD94/NKG2A and 2DL4 NK cell inhibitory receptors respectively, which will suppress the NK-mediated lysis of the donor cells. Last but not least, the investigator will evaluate the performance of engineered HVPs using in vitro and in vivo models. The investigator will examine the growth of luciferase-expressing engineered HVPs and control HVPs transplanted into immunodeficient mice injected with human T cells or NK cells.